The protein folding problem remains unsolved. Recent efforts in this field have focused on small proteins that display fast folding and are also amenable to computational molecular dynamics simulated folding. Surprisingly none of these protein folding paradigms appear to have been the subject of circular permutation studies. We have selected the Trp-cage (TC), the villin headpiece (HP36), and a well studied WW domain (Pin1) for this purpose. Our newly designed circular permutants of these proteins retain native-like structure, though the chains were, in effect, cyclized and cleaved at new locations. Circular permutation represents a powerful probe of folding pathways since it recycles the contact order of key interactions that can drive folding and thus separates fold topology from protein sequence. Our circular permutant model systems will be used to address major questions concerning protein folding dynamics and folding pathway selection. Three distinct spectroscopic techniques will be employed: NMR relaxation dynamics, and UV-resonance-Raman (UVRR) & fluorescence monitored T-jumps. By applying all of these dynamics and melting measures, monitoring the endogenous probes at numerous sites in the sequences, we should be able to distinguish between discreet (even if multiple) folding pathways and downhill folding scenarios. We anticipate deriving complete experimental folding landscapes that can be used for comparisons with computational folding studies. The specific protein folding questions that can be addressed with these model systems are: 1) contact order effects on dynamics and folding pathway selection (from all three systems), 2) serial versus all-at-once hydrophobic core formation (TC and HP36), 3) the timing of the formation of multiple specific helix/helix and helix/loop interactions in an all-alpha protein framework (HP36), and 4) pathways of -sheet formation (hairpin nucleation versus the effects longer loop conformation search times) (WW domains). PUBLIC HEALTH RELEVANCE: Protein misfolding is associated with more than 40 human diseases and conditions; these medical conditions cause human suffering and exact a tremendous societal burden. The proposed studies will yield a greater understanding of protein sequence and topology features that result in folding dynamics retardation or folding traps that can enhance misfolding. This would be a valuable addition to knowledge that could enhance both protein fold prediction from sequence, providing fundamental insights into misfolding pathways and could provide targets for therapeutic and protein engineering interventions.